Peniphenone and penilactone formation in Penicillium crustosum via

Subscriber access provided by Washington University | Libraries

Communication

Peniphenone and penilactone formation in Penicillium crustosum via 1,4-Michael additions of ortho-quinone methide from hydroxyclavatol to #-butyrolactones from crustosic acid Jie Fan, Ge Liao, Florian Kindinger, Lena Ludwig-Radtke, Wen-Bing Yin, and Shu-Ming Li J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b00110 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Peniphenone and penilactone formation in Penicillium crustosum via 1,4Michael additions of ortho-quinone methide from hydroxyclavatol to γbutyrolactones from crustosic acid Jie Fan,1,† Ge Liao,1,† Florian Kindinger,1 Lena Ludwig-Radtke,1 Wen-Bing Yin,2 and Shu-Ming Li1,* 1 Institut

für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Strasse 4, Marburg 35037, Germany 2 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Supporting Information Placeholder ABSTRACT: Penilactones A and B consist of a -butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a non-reducing PKS ClaF is responsible for the formation of clavatol and the PKSNRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to -butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a non-heme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two non-enzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the -butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

For secondary metabolite (SM) production in PRB-2, several culture conditions were tested and the extracts were analyzed on HPLC (Figure S1 in the Supplement Information (SI)). Three dominant peaks were detected in a 7 days-old PD culture (Figure S1), which were identified as 9, 10,14 and terrestric acid (11)15 after isolation and structure elucidation (See SI for details, NMR data and spectra are given in Tables S6−S10 and Figures S28−S45). The stereochemistry of 11 was confirmed by determination of its optical rotation and comparison with the published data.15 9 has not been described before and therefore was confirmed by X-ray analysis (Table S11). Two additional minor peaks were proven to be 5 and hydroxyclavatol ethyl ether (12) (Figures 1A and S1).14 A OH

HO

OH

OH

HO

OH

O

O O

O O

O OH

O

OH

OH

OH

OH

O

O O

O

O OH

OH OH

O penilactone D (4)

peniphenone D (3)

OH

O

O

OH

OH O

OH O

OH

O

O

O

O

HO

O OH

O terrestric acid (11)

B O

O

O

O

O

O

OH

OH

OH

clavatol (5)

2

OH

O

O

o-quinone methide (6) O

O

OH

HO

OH O

O

OH O

O CH3CO-SCoA

O L-malic

O

penilactone A (1)

O oxaloacetic acid

OH

OH O

O

OH

HO

HO

HO OH

O (R)-5-methyltetronic acid (7)

CO2 O

O

O

HO pyruvic acid

O

O

O O crustosic acid (13)

hydroxyclavatol ethyl ether (12)

CH3CO-SCoA

O

hydroxyclavatol methyl ether (10)

hydroxyclavatol (9)

OH

O

OH

OH

o-quinone methide (6)

clavatol (5)

OH

OH OH

O penilactone B (2)

penilactone A (1)

5 can be considered as a polyketide synthase (PKS) product. However, the responsible enzyme is unknown before. Neither the direct precursor nor the biosynthesis of 7 and 8 has been reported. Michael addition as a thermodynamically controlled 1,4-addition of active methylenes to activated olefins such as α,β-unsaturated carbonyl derivatives8 are widely used in the chemical synthesis9-12 and also involved in the biosynthesis of natural products.13 However, the substrates, enzymes and conditions for Michael addition involved in the formation of 1– 4 in nature have not been reported yet.

O

O O

Penilactones A (1) and B (2) (Figure 1A) are rare fungal metabolites and were firstly isolated from Penicillium crustosum PRB-2.1 Together with their putative precursors peniphenone D (3) and penilactone D (4) (Figure 1A), they were also identified in other Penicillium species.2-4 Feeding experiments suggested that 1 and 2 are derived from acetylCoA and L-malic acid (Figure 1B).1 It was proposed that 1 and 2 are formed by 1,4-Michael additions of two clavatol (5) molecules in its active form ortho-quinone methide (6) with a γ-butyrolactone (tetronic acid), i.e. (R)-5-methyl (7) or (S)-5carboxylmethyltetronic acid (8).1,2 This hypothesis was confirmed by a biomimetic synthesis.5-7 Acetate of hydroxyclavatol (9) instead of 5 was used at 110 °C for the synthesis. Hydroxyclavatol methyl ether (10) was also isolated from P. crustosum.3

O

O O

acid

O HO (S)-5-caboxylmethyl tetronic acid (8)

O

O OH

OH

O penilactone B (2)

Figure 1. Metabolites from PRB-2 (A) and proposed biosynthetic routes to 1 and 2 (B). 1

However, peniphenones and penilactones could only be detected in extracted ion chromatograms (EICs, data not shown). To increase their productivity, PRB-2 was cultivated in PD surface culture for 14 days. LC-MS analysis revealed clear accumulation of 1–4 (Figures 2, S1, and S2). A 30 days-old rice

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

culture also accumulated 1–4 and was therefore used for isolation and structure elucidation by MS, NMR (Tables S6–S7 and Figures S28–S31), optical rotation, and CD spectra (Figures S46−S49).1,3 The CD spectra of 1 and 2 (Figures S46 and S47) correspond very well to those reported previously.1 The stereochemistry of 3 and 4 was determined by chemical synthesis from 7 and 8 with known configuration at C-5 (Figure S21, see below for the formation of 3 from 7 and 4 from 8). Under these conditions, the production of 9 and 10 was strongly reduced. In comparison, 11 was detected as the predominant peak. Furthermore, a new peak was identified as carboxylated derivative of 11, termed crustosic acid (13) hereafter (Figures 1A, S44, and S45). 13 has an [α]20 D value of -164.1, while that of 11 at +37.1. The configuration of 13 was assigned by comparison with the optical rotation data of 5methyl- and 5-carboxylmethyltetronic acids.16

For understanding the role of 11 and 13 for 1–4, they were isolated from ∆claF mutant and fed into ∆traA mutant. Feeding 11 only restored 1 and 3 production, while 1–4 were detected after feeding with 13 (Figures 2C, S18 and S19). More interestingly, 11 was also restored after feeding with 13, but not vice versa (Figure 2D). This proved that 13 is the precursor of both 8 and 11. 11 serves then as a precursor of 7 (Scheme 1).

For biosynthetic studies on 1 and 2, the genome of PRB-2 was sequenced and the draft genome sequence was used for prediction of putative gene clusters by using AntiSMASH.17 For gene inactivation, we established a gene replacement protocol using the split marker strategy and hygromycin B as selection marker, which significantly enhances the homologous recombination events at the target gene (Figure S3).18 Based on its aromatic character, 5 is expected to be assembled by a non-reducing PKS (NR-PKS).19 One of the six NR-PKS genes pcr3094 within a 36.2 kbp large cluster (Figure 2A and Table S4) has a SAT-KS-AT-PT-ACP-MeT-TE domain structure (Abbreviations for PKS and NRPS domains as given before19,20). It shares a sequence identity of 57.7 % with CitS from Monascus ruber21 and 64.4 % with EAW12049.1 from Aspergillus clavatus (Table S4). Deletion of pcr3094, termed claF (from the clavatol cluster) hereafter, completely abolished the production of 1−5, 9, and 10 (see SI for manipulation). The two tetronic acids 11 and 13 accumulated with much higher yields in the ∆claF mutant than in PRB-2 (Figures 2B, S4, and S6). Feeding 5 to the mutant restored the production of 1–4 and 9 (Figure S15). To provide more evidence for the function of ClaF as a clavatol synthase, pcr3094 was cloned into pYH-wA-pyrG and expressed in A. nidulans.22-24 The formation of 5 in the transformant JF11 was confirmed by LC-MS (Figure S20) and 1H NMR analyses after isolation. These results proved that ClaF is responsible for 5 formation in the biosynthesis of 1–4 (Scheme 1). To identify the genetic potential for 7 and 8, we focused on PKSNRPS hybrid enzymes, because tetronic acids like carlosic acid are usually assembled by such enzymes.25 Analysis of the draft sequence revealed the presence of a candidate gene pcr11009, termed traA (from the terrestric acid cluster), within a 33.6 kbp large cluster. TraA with a domain structure KS-AT-DH-MeTKR-ACP-C-A-PCP (Figure 2A) shares a sequence identity of 69.6 % with CaaA in the carlosic acid biosynthesis (Table S5).25 Deletion of traA completely abolished the production of 1–4, indicating its involvement in the biosynthesis. As expected, 5, 9, and 10 were accumulated in the ∆traA mutant (Figures 2C and S8). Surprisingly, the production of 11 and 13 were also totally blocked. To restore the production of 1–4, we chemically synthesized 7 and 8 (Figure S21)5,7,26,27 and fed them to the ∆traA mutant. LC-MS analysis revealed that feeding 7 restored the production of 1 and 3, but not 2 and 4. In contrast, 2 and 4, but not 1 and 3 were detected in the culture of ∆traA mutant fed with 8 (Figures 2C, S16 and S17). This proved that TraA is involved in the formation of 7 and 8, which cannot be converted to each other (Scheme 1).

ACS Paragon Plus Environment

Page 2 of 7

Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society It can be concluded that 13 is the product of TraA with or without other enzymes and mainly converted to the predominant product 11 in PRB-2. Only small amounts of 11 and 13 undergo degradation to 7 and 8 for the formation of 1−4 (Figure 2D and Scheme 1).

A

11

B

4 280 nm 13

9

3 and 5 2 10

WT

1

EICs

WT 11

280 nm 13

claD

5

claD

EICs 11 280 nm

13

claF claF

EICs 11 9

280 nm 13

claJ

5 10

claJ

EICs

15

10

25

20

30

35

t/min C

13

280 nm

9

10

4 5

traH

2

traH

EICs 9

10 traA

5

280 nm

traA

EICs 3 and 5

D

10

1

9

280 nm

traA + 7 traA + 7

EICs 5

9

10

4

280 nm

2 traA + 8 traA + 8

EICs 11

10

For biochemical characterization, claD was amplified and cloned into pET28a (+). The purified ClaD (Figure S23) was used for incubation with 5 in the presence of ascorbate (AA), Fe[(NH4)2(SO4)2], and 2-oxoglutarate (2OG).28,29 HPLC analysis confirmed the oxidation of 5 to 9 with a conversion yield of 22.5 % after incubation with 2 µg protein at 37°C for 30 min (Figure 3A). Nearly no consumption of 5 was detected in the assays without ascorbate or 2-oxoglutarate. Replacing ascorbate by dithiothreitol (DTT) or without additional FeII reduced the activity significantly. These results proved that ClaD acts as a non-heme FeII/2-oxoglutarate-dependent oxygenase and oxidizes 5 to yield 9. Determination of kinetic parameters gave a KM of 0.30 mM toward 5 and a turnover number (kcat) of 0.26 s−1 (Figure 3B). To prove the conversion between 9 and 6, 9 was incubated in H2O and H218O at 25°C for 16 h. MS data in positive and negative modes confirmed the incorporation of 18O into 9 and therefore the equilibration (Figures 3C and S24).

traA + 11

1

traA + 11

EICs 13 11

280 nm

9 4

3 and 5 2 1 10

traA + 13 traA + 13

EICs

10

Regarding Michael addition, we presumed a more active intermediate than 5 for the formation of 6. Detailed inspection of the cla cluster (Figure 2A, Table S4) revealed the presence of genes coding for an oxygenase (claD) and a cytochrome P450 (claJ). ClaD comprises 338 amino acids and shares a sequence identity of 53.8 % with CitB in the citrinin biosynthesis.21 It also contains the typical conserved 2-His-1-Asp ion-binding triad (His184, His202 and Asp187) of non-heme FeII/2-oxoglutaratedependent oxygenases (Figure S22). Deletion of claD abolished the production of 1–4 and 9, whereas 5 was clearly accumulated (Figures 2B and S5). Feeding 9 in the ∆claD mutant restored the production of 1–4 (Figure S14), proving its role in the conversion of 5 to 9.

3 and 5 9

280 nm

Having the both backbone genes/enzymes identified, we intended to investigate the conversion of 13 to 8 and 11, 11 to 7, and the metabolism of 5. Inactivation of the oxygenase gene traH abolished the production of 1, 3, and 11, confirming its involvement in the decarboxylation and isomerization of 13 to 11 (Figures 2C and S13). In the deletion mutants of the cytochrome P450 traB and the dehydrogenase traD, no accumulation of 11, 13, or 1–4 was detected (Figures S9 and S10), proving their roles in the 13 formation (Scheme 1). Deletion of traE and traF did not result in significant changes in SM production (Figures S11 and S12).

15

20

25

30

35

t/min Figure 2. Schematic representation of clavatol and terrestric acid clusters in PRB-2 (A) and LC-MS results of deletion mutants (B and C) as well as of ∆traA mutant fed with putative precursors (D). EICs refer [M+H]+ ions of 1–6 and 11, 13 or [M+Na]+ of 9 and 10 with tolerance ranges of ± 0.005.

ACS Paragon Plus Environment

Journal of the American Chemical Society A

280 nm

C

5 22.5 %

9

i

+AA, FeII, 2OG

ii 4.3 %

O

OH O2

6.5 %

v

standard 5

viii

standard 9

10

15

20

[M+Na] : 219.0628 [M-H]-: 195.0663

7+9 in H2O

1

3+9 in H2O

4

9

8

1 3

9

+

8+9 in H2O 9 4

H 2O

2 O

25

OH

O

OH

4

CH218OH H218O

40

OH

3

2

4+9 in H2O standard 1 standard 2 standard 3 standard 4

1

O

CH3

30

Km=0.30 mM kcat=0.26 s-1

20 10 0 0.0

9

CH2OH

OH OH Fe2+ CH3 2-oxoglutarate succinate hydroxyclavatol (9) clavatol (5)

t/min

50

OH

CO2

+DTT, FeII, 2OG denat. enzyme +AA, FeII, 2OG

vii

7

ClaD

+AA, FeII

vi

B

O

+AA, 2OG

iv

3

A

+FeII, 2OG

iii

V [nmol*mg-1*min-1]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 7

0.5

1.0

1.5

18

[ O]-hydroxyclavatol (9) [M+Na]+: 221.0670 [M-H]-: 197.0705

o-quinone methide (6)

8

9

7

[M+H]+: 179.0703 [M-H]-: 177.0557

standard 7 standard 8 standard 9

2.0

concentration of 5 [mM]

Figure 3. Functional proof of ClaD as a non-heme FeII/2-oxoglutaratedependent clavatol oxidase (A and B) and determination of the equilibration between 9 and 6 (C).

ClaJ shares clear sequence homology with fungal cytochrome P450 enzymes, e.g. 42.0 % identity with BAJ04372.1 from Aspergillus oryzae.30 Deletion of claJ resulted in the abolishment of 1−4 (Figures 2B and S7), but still retained the production of 5, 9, 11, and 13. This indicates its role in the C-C double bond cleavage of 11 and 13 (Scheme 1). However, ClaJ could also catalyzes the connection of the two building blocks via Michael addition.

0

5

10

15

t/min

20

25

30

35

B O

O

O H

O

3

H

HO 7

O

4 O

H O

OH

O

H O

O O O

O

O

O

O

3 HO

OH 8´

O

OHOH

H 2O O

OH

O

OH

O H

O

OH

OH O 6 O

O

OH O

O H

H 2O O

H O O H

O

OH O

H

6 O OH

HO

O

O

O O

O H

O HO 8

COOH O

OH O

OH 8´

OH

O

H O

O

OH

O O HOOC H

OH O

O

O

H

O OH

O

O O

O

O COOH

H O O

O COOH

OH

O

OHOH 4 O

O

O

OH

COOH

HO

H O

OH O

O

O 4 O

H

O

H

H 2O O

3

H

O

O H

O

O OH

1 O

O OH

HO OH

H H 2O O

OH

OH O 6 O

O

O

OH O

COOH

COOH

H

6

O

O OH

HO OH O 2

O

O

HO OH

O OH

COOH

OH O

O OH

O

O

H

O H

H O

OH

O

O COOH

OH O

O OH O O COOH

Figure 4. Non-enzymatic formation of penilactones and peniphenone. (A) LC-MS analysis of 48 h-incubation mixtures. Absorptions at 254 nm (1−4, 9) or EICs (7 and 8) are illustrated. (B) Proposed mechanism of non-enzymatic formation of 1−4 via Michael addition.

ACS Paragon Plus Environment

Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

ClaF

ClaF

O SAT KS

AT

PT ACP MeT TE

S

SAT KS

AT

O

OH

PT ACP MeT TE

S

S

O

OH O

O

O

O

+H2O

3 x CO2, 3 x HSCoA 2 x S-adenosylhomoserine

3 x malonylSCoA 2 x SAM

O

OH

OH

O

OH

OCH3

hydroxyclavatol methyl ether (10)

hydroxyclavatol (9)

clavatol (5)

O

O

O

OH OH

O O

non-enzymatic conversions

OH

ClaD

-H2O spontaneous

O

OH

-CH3OH +H2O

+CH3OH

OH OC2H5

hydroxyclavatol (9) -H2O

OH

-C2H5OH

hydroxyclavatol ethyl ether (12)

O

O

+C2H5OH

OH

OH

OH

O

o-quinone methide (6)

o-quinone methide (6) TraB, TraD O

TraA KS

AT

MeT DH

S O

KR O O

3 x malonylSCoA

ACP C

ClaJ

O

A

S

PCP

HOOC

O L-malic

HO

(S)-5-carboxylmethyltetronic acid (8)

acid

6

O

HO OH

O

OH O

O OH OH

non-enzymatic Michael addition

HOOC

crustosic acid (13)

S

OH

O

HO

O

O

O O

O

COOH

O

non-enzymatic Michael addition

penilactone D (4)

O OH COOH

penilactone B (2)

TraH

O

O O

3 x CO2 3 x HSCoA

6

6

OH

O

O

terrestric acid (11)

O

O

ClaJ

O

HO

O

OH

O

OH OH

O

non-enzymatic Michael addition

peniphenone D (3)

(R)-5-methyltetronic acid (7)

OH

O O

non-enzymatic Michael addition

OH

HO

O

O OH

penilactone A (1)

Scheme 1. Proposed biosynthetic pathways of penilactones and peniphenones in P. crustosum. For preparing feeding experiments in ∆claJ, we carried out control incubations of 7 with 9 and 8 with 9 in water at 25°C, which delivered surprising results, i.e. the non-enzymatic Michael addition under these mild conditions (Figure 4A). In the first combination, 3 was detected as the major and 1 as a minor product, while 4 as the major and 2 as a minor product in the case of 8 with 9. When 3 and 4 were incubated with 9, 1 and 2 were detected (Figure 4A). Formation of 3 and 4 is timeand pH-dependent (Figures S25 and S26). 3 and 4 are formed under neutral or acidic conditions. When pH values were higher than 5.0, diclavatol3 was also detected (Figure S26). It is obvious that the active intermediate 6 can be easily formed from 9 in aqueous system and initiates the Michael additions (Figure 4B), which was confirmed by incubation of 9, 10, and 12 in different solvents. They are stable in acetonitrile. Alcohols determined the end products of 6 (Figure S27, Scheme 1). All these results indicate that ClaJ is likely not involved in the Michael addition, probably in the conversion of 11 to 7 and 13 to 8 (Scheme 1). Taken together, 1–4 are formed by enzymes from independent pathways of two separate gene clusters (Scheme 1). The tra cluster assembles 13, which is converted to 11. Both acids deliver the two γ-butyrolactones 7 and 8. The cla cluster provides the highly active 6 by a spontaneous dehydration of 9. This initiates the two step non-enzymatic Michael additions by the intermolecular nucleophile attacking of 6 to 7 or 8 and subsequent reaction with 3 and 4. Thus, this study provides an excellent example for SM with complex structures that are formed by enzymes from different pathways and by combination of enzymatic and non-enzymatic reactions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials, experimental procedures, physiochemical properties and spectroscopic data (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

ORCID Shu-Ming Li: 0000-0003-4583-2655 Wen-Bing Yin: 0000-0002-9184-3198 Author Contributions †These

authors contributed equally to this work.

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Tianjiao Zhu (Ocean University of China, Qingdao) for providing strain PRB-2, Rixa Kraut, Stefan Newel, and Andreas Heine (University of Marburg) for taking MS, NMR spectra and X-ray crystal analysis, respectively. This project was financially funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) − Li844/11-1 and INST 160/620-1 as well as the National Natural Science Foundation of China − 31861133004. Jie Fan (201507565006) and Ge Liao (201607565014) are scholarship recipients from the China Scholarship Council.

REFERENCES (1) Wu, G.; Ma, H.; Zhu, T.; Li, J.; Gu, Q.; Li, D. Penilactones A and B, two novel polyketides from Antarctic deep-sea derived fungus Penicillium crustosum PRB-2. Tetrahydron 2012, 68, 9745. (2) Li, H.; Jiang, J.; Liu, Z.; Lin, S.; Xia, G.; Xia, X.; Ding, B.; He, L.; Lu, Y.; She, Z. Peniphenones A-D from the mangrove fungus Penicillium dipodomyicola HN4-3A as inhibitors of Mycobacterium tuberculosis phosphatase MptpB. J. Nat. Prod. 2014, 77, 800. (3) Wu, G. Studies on secondary metabolites of three different

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

marine environment-derived fungi: structures and bioactivities. Dissertation, Ocean University of China 2014, (4) Sun, W.; Chen, X.; Tong, Q.; Zhu, H.; He, Y.; Lei, L.; Xue, Y.; Yao, G.; Luo, Z.; Wang, J.; Li, H.; Zhang, Y. Novel small molecule 11beta-HSD1 inhibitor from the endophytic fungus Penicillium commune. Sci. Rep. 2016, 6, 26418. (5) Spence, J. T. and George, J. H. Biomimetic total synthesis of ent-penilactone A and penilactone B. Org. Lett. 2013, 15, 3891. (6) Pantin, M.; Brimble, M. A.; Furkert, D. P. Total synthesis of (-)-peniphenone A. J. Org. Chem. 2018, 83, 7049. (7) Spence, J. T. and George, J. H. Total synthesis of peniphenones A-D via biomimetic reactions of a common oquinone methide intermediate. Org. Lett. 2015, 17, 5970. (8) Tokoroyama, T. Discovery of the Michael reaction. Eur. J. Org. Chem. 2010, 2009. (9) Wadhwa, P.; Kharbanda, A.; Sharma, A. Thia-Michael addition: An emerging strategy in organic synthesis. Asian J. Org. Chem. 2018, 7, 634. (10) Mather, B. D.; Viswanathan, K.; Miller, K. M.; Long, T. E. Michael addition reactions in macromolecular design for emerging technologies. Prog. Polym. Sci. 2006, 31, 487. (11) Zhang, Y. and Wang, W. Recent advances in organocatalytic asymmetric Michael reactions. Catal. Sci. Technol. 2012, 2, 42. (12) Nising, C. F. and Bräse, S. The oxa-Michael reaction: from recent developments to applications in natural product synthesis. Chem. Soc. Rev. 2008, 37, 1218. (13) Miyanaga, A. Michael additions in polyketide biosynthesis. Nat. Prod. Rep. 2019, DOI: 10.1039/C8NP00071A. (14) Astudillo, L.; Schmeda-Hirschmann, G.; Soto, R.; Sandoval, C.; Afonso, C.; Gonzalez, M. J.; Kijjoa, A. Acetophenone derivatives from Chilean isolate of Trichoderma pseudokoningii Rifai. World J. Microbiol. Biotechnol. 2000, 585. (15) Nukina, M. Terrestric acid as a phytotoxic metabolite from Pyricularia oryzae Cavara. Agric. Biol. Chem. 1988, 52, 2357. (16) Clutterbuck, P. W.; Haworth, W. N.; Raistrick, H.; Smith, G.; Stacey, M. Studies in the biochemistry of micro-organisms: The metabolic products of Penicillium charlesii G. Smith. Biochem. J. 1934, 28, 94. (17) Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruccoleri, R.; Lee, S. Y.; Fischbach, M. A.; Müller, R.; Wohlleben, W.; Breitling, R.; Takano, E.; Medema, M. H. antiSMASH 3.0 - a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015, 43, W237. (18) Goswami, R. S. Targeted gene replacement in fungi using a split-marker approach. Methods Mol. Biol. 2012, 835, 255.

(19) Cox, R. J. Polyketides, proteins and genes in fungi: programmed nano-machines begin to reveal their secrets. Org. Biomol. Chem. 2007, 5, 2010. (20) Miyanaga, A.; Kudo, F.; Eguchi, T. Protein-protein interactions in polyketide synthase-nonribosomal peptide synthetase hybrid assembly lines. Nat. Prod. Rep. 2018, 35, 1185. (21) He, Y. and Cox, R. J. The molecular steps of citrinin biosynthesis in fungi. Chem. Sci. 2016, 7, 2119. (22) Yin, W. B.; Chooi, Y. H.; Smith, A. R.; Cacho, R. A.; Hu, Y.; White, T. C.; Tang, Y. Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth. Biol. 2013, 2, 629. (23) Li, W.; Fan, A.; Wang, L.; Zhang, P.; Liu, Z.; An, Z.; Yin, W.B. Asperphenamate biosynthesis reveals a novel two-module NRPS system to synthesize amino acid esters in fungi. Chem. Sci. 2018, 9, 2589. (24) Chiang, Y. M.; Ahuja, M.; Oakley, C. E.; Entwistle, R.; Asokan, A.; Zutz, C.; Wang, C. C.; Oakley, B. R. Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of aspercryptin. Angew. Chem. Int. Ed. Engl. 2016, 55, 1662. (25) Yang, X. L.; Awakawa, T.; Wakimoto, T.; Abe, I. Three acyltetronic acid derivatives: noncanonical cryptic polyketides from Aspergillus niger identified by genome mining. Chembiochem. 2014, 15, 1578. (26) Adrian, J. and Stark, C. B. Total synthesis of muricadienin, the putative key precursor in the solamin biosynthesis. Org. Lett. 2014, 16, 5886. (27) Stebbins, N. D.; Yu, W.; Uhrich, K. E. Enzymatic polymerization of an ibuprofen-containing monomer and subsequent drug release. Macromol. Biosci. 2015, 15, 1115. (28) Ran, H.; Wohlgemuth, V.; Xie, X.; Li, S.-M. A non-heme FeII/2-oxoglutarate-dependent oxygenase catalyzes a double bond migration within a dimethylallyl moiety accompanied by hydroxylation. ACS Chem. Biol. 2018, 13, 2949. (29) Steffan, N.; Grundmann, A.; Afiyatullov, A.; Ruan, H.; Li, S.M. FtmOx1, a non heme Fe(II) and alpha-ketoglutaratedependent dioxygenase, catalyses the endoperoxide formation of verruculogen in Aspergillus fumigatus. Org. Biomol. Chem. 2009, 7, 4082. (30) Nazir, K. H. M. N. H.; Ichinose, H.; Wariishi, H. Molecular characterization and isolation of cytochrome P450 genes from the filamentous fungus Aspergillus oryzae. Arch. Microbiol. 2010, 192, 395.

ACS Paragon Plus Environment

Page 6 of 7

Page 7 of 7 Table of Contents

MeT ACP

AT KS

DH

KR

A C

O

PCP

OH

HO O

O

O

O

O

HOOC

HO O

O

O

crustosic acid O

O

O

OH

O OH

OH

OH

OH

2

spontaneous

hydroxyclavatol

clavatol

-butyrolactones

O

OH O O

O

R

terrestric acid

OH

O

O R=CH3 or CH2COOH

OH

spontaneous Michael addition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

ortho-quinone methide

SAT KS AT PT ACP MeT TE

penilactone A O O

HO OH

OH O

O

O OH COOH

penilactone B

ACS Paragon Plus Environment

7